Use of prebiotics for preventing or treating oxidation stress

ABSTRACT

The invention relates to the use of prebiotics for the preparation of food preparations, functional foods, or pharmaceutical compositions for preventing or treating oxidative stress in particular linked to the consumption of fructose. The invention also relates to a food preparation comprising simple carbohydrates, in particular fructose, in combination with prebiotics.

A subject of the present invention is the use of prebiotics for thepreparation of food preparations, functional foods, or pharmaceuticalcompositions intended to prevent or treat oxidative stress.

Oxidative stress is the result of an imbalance in the organism in favorof pro-oxidant species relative to anti-oxidant species.

Pro-oxidant species are generally free radicals and in particularoxygenated free radicals. The presence of an unpaired electron makesthese compounds extremely reactive vis-à-vis the biologicalmacromolecules of the organism; lipids, carbohydrates, proteins andnucleic acids are thus preferred targets of these species. The oxidativedegradation of these macromolecules leads to many cellular malfunctions.

The origin of these free radicals is essentially found in the metabolismof oxygen. Most of the oxidative stress originates from the energymetabolism. The final stage of the oxidation of food, namely themitochondrial respiratory chain, is thus the origin of the formation ofoxygenated free radicals. Moreover, during inflammatory reaction, thestimulation of the phagocytes is also accompanied by the formation offree radicals.

The anti-oxidant defenses of the organism use protein systems, such assuperoxide dismutase, but also anti-oxidant compounds provided by food,such as vitamins C and E, or other nutrients, such as carotenoids,polyphenols or flavonoids.

Dietary imbalances can be the origin of oxidative stress. The inventorshave in particular previously shown that food which is too rich insugars, and in particular in saccharose (Busserolles et al., 2002a) andfructose (Busserolles et al., 2002b), could cause significant oxidativestress. These pro-oxidant effects are even more significant the lowerthe content of anti-oxidants in the food is.

Fructose is a monosaccharide, the consumption of which has greatlyincreased, either as it is or in the form of saccharose. Because oftheir low production costs, syrups that are rich in fructose producedfrom corn are preferably used in sugary drinks. While fructose isnaturally present in honey and in fruits where it is associated withmany protective micronutrients, the consequences of an unrestrictedincrease of this carbohydrate in purified form on health arequestionable. In fact, fructose has many properties which distinguish itfrom other sugars and the high intake of this carbohydrate could beresponsible for undesirable metabolic effects.

The modalities for combating oxidative stress generally include the useof anti-radical nutrients having a direct effect on free radicals:carotenes, ascorbic acid (vitamin C), tocopherols (vitamin E),polyphenols (U.S. Pat. No. 6,207,702).

The present invention results from the inventors demonstrating thatprebiotics and more particularly fructooligosaccharides (FOS) can combatthe oxidative stress resulting from an excess of fructose in food.

Prebiotics are non-digestible complex carbohydrates broken down by themicroorganisms of the intestinal flora and whose breakdown hasbeneficial health effects for the host. These microorganisms aregenerally bacteria, and in particular bifidobacteria, which areessentially found in the colon. The beneficial effects provided by saidmicroorganisms can be due to the selective stimulation of the growth ofcertain species of microorganisms, in particular bifidobacteria, and/orto the release of metabolites originating from the conversion of theprebiotics by the microorganisms.

At present, the only clearly defined prebiotics are sugar polymers witha degree of polymerization comprised between 2 and 12, classified ascomplex carbohydrates: oligosaccharides. Thus, apart fromfructooligosaccharides whose effects are most documented, fructans,galactooligosaccharides, xylooligosaccharides, soybean oligosaccharides,gentiooligosaccharides or also isomaltooligosaccharides, may bementioned.

Fructooligosaccharides (FOS) are obtained either by hydrolysis ofinulin, or by enzymatic synthesis, by transfructosylation fromsaccharide precursors. They correspond to the general formulaGlucosyl-(Fructosyl)_(n)-Fructose or (Fructosyl)_(m)-Fructose where nrepresents an integer from 1 to 8 and m represents an integer from 1 to8. In most cases FOS preparations are not homogeneous. They comprisemixtures of chains of variable size. Moreover, in the case of thepreparation of FOS by enzymatic synthesis, the polymers correspond tothe formula Glucosyl-(Fructosyl)_(n)-Fructose (1≦n≦8), while the FOSprepared by hydrolysis correspond to the two formulaeGlucosyl-(Fructosyl)_(n)-Fructose and/or (Fructosyl)_(m)-Fructose (1≦n≦8and 1≦m≦8). The FOS in particular comprise short-chainfructooligosaccharides, synthesized by transfructosylation, whose degreeof polymerization is less than 6, and in particular short-chain FOS with2, 3 or 4 fructose units such as 1-kestose, nystose andfructosyl-nystose.

Fructans are polymers in which the fructosyl-fructose type bondspredominate.

The galactooligosaccharides are formed by 2 to 6 hexose units, theymainly comprise galactose as a base unit. They are synthesized by theaction of β-galactosidase on lactose.

Xylooligosaccharides originate from the hydrolysis of xylan, they areconstituted by xylose.

Soybean oligosaccharides are extracted from soybean, these are mainlyoligosaccharide mixtures comprising from 1 to 4 osidic units, the mainconstituents being raffinose and stachyose.

Gentiooligosaccharides are polymers originating from the digestion ofstarch, in which most of the bond has theβ-glucopyranosyl-(1→6)-glucopyranose form.

Isomaltosaccharides are also glucose polymers which originate from thehydrolysis of starch, these are mixtures of isomaltose, panose,isomaltotriose and other branched polymers containing 4 or 5 glucoseunits.

The inventors have shown that the addition of prebiotics to the foodintake, advantageously the addition of FOS, allowed a reduction inoxidative stress due in particular to a diet rich in sugars, and inparticular fructose.

The purpose of the invention is to provide new means for preventing ortreating oxidative stress.

A subject of the invention is the use of prebiotics for the preparationof food preparations, functional foods, or pharmaceutical compositionsintended to prevent or treat oxidative stress.

A subject of the invention is more particularly the above-mentioned useof at least one oligosaccharide chosen from:

-   -   fructanes    -   fructooligosaccharides (FOS)    -   galactooligosaccharides    -   xylooligosaccharides    -   soybean oligosaccharides    -   gentiooligosaccharides    -   isomaltooligosaccharides

as defined above.

The invention more particularly relates to the above-mentioned use offructooligosaccharides (FOS) of general formulaGlucosyl-(Fructosyl)_(n)-Fructose or (Fructosyl)_(m)-Fructose where nrepresents an integer from 1 to 8, in particular from 1 to 5, and mrepresents an integer from 1 to 8, in particular from 1 to 5, such asshort-chain FOS, 1-kestose, nystose or fructosyl-nystose.

A subject of the invention is also the use of prebiotics in the contextof the prevention or treatment of oxidative stress linked to theconsumption of sugars.

The invention more particularly relates to the use of prebiotics in thecontext of the prevention or treatment of oxidative stress linked to theconsumption of fructose.

The invention in particular relates to the use of prebiotics in thecontext of the prevention or treatment of oxidative stress due to aconsumption of fructose in food greater than approximately 50 g/day onaverage.

The invention also relates to the use of prebiotics, where saidprebiotics are administered at a daily dose of approximately 1 g toapproximately 20 g, in particular approximately 2 g to approximately 17g, in particular approximately 5 g to approximately 15 g.

A subject of the invention is also the use of prebiotics as compoundswith an anti-radical effect in the context of the prevention ortreatment of oxidative stress.

A subject of the invention is also the use of prebiotics as compoundswith an anti-aging effect linked to an effect which protects the cellsof the organism against the action of free radicals.

The invention also relates to any food preparation comprising simplecarbohydrates in combination with prebiotics.

The invention more particularly relates to a food preparationcomprising:

-   -   at least one simple carbohydrate such as fructose or saccharose,    -   in combination with one or more oligosaccharides chosen from:        -   fructanes        -   fructooligosaccharides (FOS)        -   galactooligosaccharides        -   xylooligosaccharides        -   soybean oligosaccharides        -   gentiooligosaccharides        -   isomaltooligosaccharides    -   as defined above.

Advantageously the food preparation of the invention is such that theproportion of prebiotics represents at least 5% by weight of thequantity of simple carbohydrates present in said preparation.

The invention in particular relates to a food preparation in which theproportion by weight of fructooligosaccharides (FOS) relative to thequantity of fructose present in said preparation varies between 10% and100% and is in particular approximately 15% to approximately 35% and isin particular approximately 20%.

The invention in particular relates to a food preparation comprising amixture of fructooligosaccharides (FOS), as defined above, comprising64% Glucosyl-(Fructosyl)_(n)-Fructose and 36% (Fructosyl)_(m)-Fructosewith average degrees of polymerization of 4.8.

The invention more particularly relates to a food preparation comprisinga mixture of fructooligosaccharides (FOS), as defined above, comprising64% Glucosyl-(Fructosyl)_(n)-Fructose and 36% (Fructosyl)_(m)-Fructose,with average degrees of polymerization of 4.8, the proportion by weightof said FOS present in said preparation varying between 10% and 100%,and in particular being approximately 15% to approximately 35%,preferably approximately 20%, relative to the quantity of fructosepresent in said preparation.

According to a preferred embodiment, the FOS mixture used corresponds tothe Raftilose® P₉₅ preparation from ORAFTI, Thienen, Belgium.

A subject of the invention is also a food product containing the foodpreparation defined above, said food product being chosen from a groupcomprising pastries, confectionary, desserts, drinks, cereal bars,chocolate bars, sweet bars, breakfast cereals, dairy products and foodsupplements.

DESCRIPTION OF THE INVENTION

The inventors have shown, in an animal model, that the addition offructooligosaccharides (FOS) to the food intake allows a reduction inoxidative stress due to a diet enriched in fructose.

40 weaned male rats of the Wistar-Han type (IFFA-CREDO; L'Arbresle,France) 6 week old and weighing approximately 150 g were used. The ratswere placed in cages with a wire mesh back in a temperature-controlledroom (22° C.) with day/night cycles of 12 hours. The animals weretreated according to the recommendations of the INRA Ethics Committee,decree No. 87-848.

First of all the rats were fed following a semi-purified starch-baseddiet for 7 days: They were then randomly divided into 4 groups of 10rats: one starch group (A), one fructose group (F), one starch+FOS group(A/FOS) and one fructose+FOS group (F/FOS). They then followed theirappropriate diet for 4 weeks.

The food and distilled water were provided ad libitum. The compositionof the food rations was as follows (in g/kg): Group Group Group Group AF A/FOS F/FOS Starch 650 — 550 — Fructose — 650 — 550 FOS (Raftilose ®P₉₅) — — 100 100 Casein 200 200 200 200 Corn oil 50 50 50 50 Alphacel 5050 50 50 Methionine D, L 3 3 3 3 Choline bitartrate 2 2 2 2 Mineralmixture (AIN-76) 35 35 35 35 Vitamin mixture (AIN-76A) 10 10 10 10

The AIN-76 and AIN-76A mixtures were provided by ICN Biomedicals, Orsay,France.

The FOS (Raftilose® P₉₅) were obtained from ORAFTI, Thienen, Belgium.They were introduced into the food gradually in order to avoid diarrhoeawhich could occur in response to too rapid an administration of largequantities of this compound. Raftilose® P₉₅ is a mixture ofGlucosyl-(Fructosyl)_(n)-Fructose (64%) and of (Fructosyl)_(m)-Fructose(36%) with average degrees of polymerization of 4.8.

4 days before sacrifice, the animals were housed individually instainless steel cages with ad libitum access to the water and food.Urine-samples were recovered 24 hours before sacrifice in 50 mlgraduated tubes, the volumes were precisely measured, the samples werethen centrifuged and kept at −80° C. until analysis. At the time ofsacrifice the rats were weighed, then anesthetized using sodiumpentobarbital (intra-peritoneal injection at 40 mg/kg) and killed. Theblood was taken from the abdominal aorta and placed in heparinizedtubes. The plasma obtained after centrifugation at low speed (2000 g, 15min) was kept at −80° C. for the biochemical analyses. The heart wasrapidly removed then washed in an ice-cooled saline Solution (NaCl 9g/l), placed in liquid nitrogen and kept at −80° C.

Two types of measurements well known to a person skilled in the art werethen taken in order to determine the intensity of oxidative stress ofthe animals as a function of their diet: a measurement of the substancesreactive to thiobarbituric acid (TBARS) and a measurement of the ratioof the plasma concentrations of vitamin E and triglycerides.

A statistical analysis of the results was carried out using the Statviewprogram (Abacus Concepts Inc., Berkeley, Calif.). The data wereexpressed as the average of the results obtained for the 10 animals ofeach food group±standard deviation. The analysis of the variance (ANOVA;P<0.05) was used in order to determine the main effects (sugar and FOS)and their interactions. The differences were considered to besignificant when p<0.05.

These results indicate that the animals following the fructose diet aresubjected to a significantly greater oxidative stress than that of thecontrol animals (subjected to the starch diet) and that the addition ofFOS allows significant reduction in the oxidative stress linked to theconsumption of fructose.

EXAMPLE 1

Measurement of the Substances Reactive to Thiobarbituric Acid (TBARS)

Measurement of TBARS allows evaluation of the level of lipidperoxidation of a sample subjected to an oxidative stress. The greaterthe value of TBARS the higher the level of oxidative stress.

The levels of plasma TBARS were determined by spectrofluorometry on anLS 5 device (Perkin Elmer, Norwalk, Conn., USA). A method adapted fromOkhawa et al. (1979) was used as previously described (Rayssiguier etal., 1993). The level of the urinary TBARS was measured as described inLee et al. (1992) and calculated on the basis of a urinary volume of 24hours. Finally, the measurement of the heart TBARS was based on Ohkawaet al. (1979), they allow the evaluation of the susceptibility of theheart lipids to peroxidation. The heart tissues were homogenized on iceat a ratio of 1 g of fresh tissue to 9 ml of KCl 150 mmol/l using aPolytron homogenizer, these homogenates were then subjected to a lipidperoxidation induced by a FeSO₄ (2 μmol/l)-ascorbate (50 μmol/l) mixturefor 30 minutes in a bath at 37° C. in the absence of oxygen, a1,1,3,3-tetraethoxypropane control was used; the TBARS were thenmeasured by spectrophotometry (Uvikon 941 plus series, KontronInstruments, St Quentin en Yvelines, France).

The results obtained are shown in the following table: Diet Anova^(a)Starch Fructose Starch + FOS Fructose + FOS Sugar FOS Sugar × FOS PlasmaTBARS  1.94 ± 0.03  2.14 ± 0.07  1.84 ± 0.02  1.96 ± 0.04 <0.01 <0.01 NSnmol/ml Urinary TBARS 11.99 ± 0.50 21.97 ± 1.58 13.40 ± 0.53 15.86 ±1.19 <0.001 <0.05 <0.001 nmol/24 h Heart TBARS 64.9 ± 4.1 98.8 ± 6.573.1 ± 3.5 83.5 ± 4.7 <0.001 NS <0.05 nmol/g of fresh weightThe results are the averages calculated for 10 animals ± standarddeviation.^(a)value of p for the ANOVA. The results of the ANOVA are significantfor p < 0.05, NS. not significant

The results indicate that the plasma, urinary and heart TBARS aresignificantly higher for the Fructose group than for the Starch group.The consumption of fructose is therefore responsible for greateroxidative stress than that which is due to the consumption of starch.

Moreover, the TBARS of the Fructose+FOS group are significantly lowerthan those of the Fructose group and are not significantly different tothose of the Starch+FOS group. The FOS therefore allow the oxidativestress which is due to the consumption of fructose to be limited.

EXAMPLE 2

Measurement of the Plasma Ratio of Vitamin E and Triglycerides

The ratio vitamin E/plasma triglycerides reflects the oxidative stressto which an organism has been subjected. The smaller the value of thisratio the greater the level of oxidative stress.

Measurement of the plasma triglyceride concentrations was carried outusing enzymatic methods according to the recommendations of the supplier(Biotrol, Paris, France). A polyvalent control serum (Biotrol-33-plus)was treated at the same time as the samples in order to check theprecision of the results of the plasma analysis.

The plasma concentrations of vitamin E were determined by reversed-phasehigh performance liquid chromatography on a Kontron series 400 device(Kontron St Quentin en Yvelines, France) using a hexane extract.α-tocopherol acetate (Sigma) was added to the samples as internalcontrol. The samples were extracted twice with hexane afterprecipitation of the proteins with ethanol. The extract was dried undernitrogen, dissolved in an ethanol-methylene chloride mixture (65:35,v/v) and injected onto a C₁₈ column (Nucleosil; 250 mm long, i.d. 46mm., 5 μm particles). Pure methanol allowed elution of the α-tocopherolin 5 minutes and the tocopherol acetate in 6.3 minutes at a flow rate of2 ml/min. The compounds were detected by UV (292 nm) then quantifiedwith internal and external calibrations using control solutions.

The obtained results are shown in the following table: Diet Anova^(a)Starch Fructose Starch + FOS Fructose + FOS Sugar FOS Sugar × FOSTriglycerides (TG) 1.76 ± 0.21 3.73 ± 0.45 1.47 ± 0.11 2.49 ± 0.26<0.001 <0.05 NS nmol/ml Vitamin E 9.01 ± 0.54 9.74 ± 0.92 7.21 ± 0.358.74 ± 0.62 NS <0.05 NS μg/ml Vitamin E/TG 5.98 ± 0.93 2.68 ± 0.12 5.03± 0.29 3.95 ± 0.61 <0.001 NS NS μg/mol TGThe results are the averages calculated for 10 animals ± standarddeviation.^(a)value of p for the ANOVA. The results of the ANOVA are significantfor p < 0.05, NS. not significant

In contrast to the starch diet, the diet rich in fructose lowers theratio Vitamin E/TG, which proves the existence of oxidative stress.

The supplementation with FOS prevents the lowering of this ratio, inother words reduces the oxidative stress which results from consumptionof a diet rich in fructose.

REFERENCES

Busserolles J., Rock E., Gueux E., Mazur A., Grolier P. and RayssiguierY., 2002a. Short term consumption of a high sucrose diet has apro-oxydant effect in rats. Brit. J. Nutr., 87(4): 337-342.

Busserolles J., Gueux E., Rock E., Mazur A. and Rayssiguier Y., 2002b.Substituting honey for refined carbohydrates protects against thepro-oxydant effect of a high fructose diet. J. Nutr., 132(11): 3379-82.

Lee H. S., Shoeman D. W. and Csallany A. S., 1992. Urinary response toin vivo lipid peroxydation induced by vitamin E deficiency. Lipids, 27:124-128.

Okhawa H., Ohishi N. and Yagi K., 1979. Assay for lipid peroxides inanimal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351-358.

Rayssiguier Y., Gueux E., Bussiere L., and Mazur A., 1993. Copperdeficiency increases the suceptibility of lipoproteins and tissues toperoxidation in rats. J. Nutr. 123: 1343-1348.

1. A method of treatment for preventing or treating oxidative stress,comprising the use of prebiotics for the preparation of foodpreparations, functional foods, or pharmaceutical compositions.
 2. Themethod of claim 1, comprising the use of at least one oligosaccharidechosen from: fructans fructooligosaceharides (FOS)galactooligosaccharides xylooligosaccharides soybean oligosaccharidesgentiooligosaccharides isomaltooligosaccharides
 3. The method of claim1, comprising the use of fructooligosaccharides (FOS) of general formulaGlucosyl-(Fructosyl)_(n)-Fructose or (Fructosyl)_(m)-Fructose where nrepresents an integer from 1 to 8, in particular from 1 to 5, and mrepresents an integer from 1 to 8, in particular from 1 to 5, such asthe short-chain FOS, 1 -kestose, nystose or fructosylnystose.
 4. Themethod of claim 1, in the context of the prevention or treatment ofoxidative stress linked to the consumption of sugars.
 5. The method ofclaim 1, in the context of the prevention or treatment of oxidativestress linked to the consumption of fructose.
 6. The method of claim 1,in the context of the prevention or treatment of oxidative stress whichis due to a consumption of fructose in food greater than approximately50 g/day on average.
 7. The method of claim 1, in which said prebioticsare administered at a daily dose of approximately 1 g to approximately20 g, in particular approximately 2 g to approximately 17 g, inparticular approximately 5 g to approximately 15 g.
 8. The method ofclaim 1, as compounds with an anti-radical effect in the context of theprevention or treatment of oxidative stress.
 9. The method of claim 1,as compounds with an anti-ageing effect linked to an effect whichprotects the cells of the organism against the action of free radicals.10. A food preparation comprising a mixture of fructooligosaccharides(FOS), as defined in claim 3, comprising 64%Glucosyl-(Fructosyl)_(n)-Fructose and 36% (Fructosyl)_(m)-Fructose, withaverage degrees of polymerization of 4.8, the proportion by weight ofsaid FOS present in said preparation varying between 10% and 100%, andin particular being approximately 15% to approximately 35%, preferablyapproximately 20%, relative to the quantity of fructose present in saidpreparation.